Biofilm Populations Research Articles

Bacterial biofilm inactivation by plasma activated nanobubble water

This research developed novel plasma activated nanobubble water (PANBW) by integrating atmospheric cold plasma and nanobubble water (NBW) technologies. Mixing of plasma reactive species with NBW to generate the PANBW makes it an effective solution for microbial biofilm inactivation and water treatment, possibly by leveraging the benefits of both technologies. Selected properties of PANBW, including the concentrations of reactive oxygen and nitrogen species (RONS) were characterized, and the stability of RONS during storage for 7 days were evaluated. The combination of argon and air as feed gases was used to determine the influence of feed gas type on RONS production and the effect of the generated PANBW on biofilm reduction. The effectiveness of PANBW in inactivating mixed-species bacterial biofilms was assessed against NBW, plasma activated water (PAW), and their combinations. This comparison involved treating biofilms of Salmonella enterica Typhimurium ATCC 13311 and Aeromonas australiensis, that were grown on stainless steel coupons by these solutions. The PANBW treatment was most effective in the inactivation of the tested mixed species biofilms with a reduction of >2 log CFU/cm2 in the biofilm population. The confocal laser scanning microscopy analysis was consistent with the bacterial inactivation results. This study highlights the potential of atmospheric cold plasma when combined with nanobubble technology, as a novel and efficient method for biofilm control and food safety applications.

Treatment of table olives with 1-methylcyclopropene, heat shock, and dimethyl dicarbonate impacts the microbial fermentation profile and composition of the fruit biofilm

In this study, we used an experimental design to assess the influence of 1-methylcyclopropene (1-MCP), a mild heat shock, and dimethyl dicarbonate (DMDC) on microbial evolution in brines and the biofilm composition of Spanish-style and naturally fermented table olives. Data were statistically analyzed using classical and compositional data analysis (CoDA). In the brines, the natural processing style raised the presence of yeast/fungi, while heat shock increased the populations of lactic acid bacteria populations. The interaction among heat shock, natural fermentation, and DMDC favored the survival of Enterobacteriaceae. Concerning biofilms, the metataxonomic analysis showed that the five most frequently detected bacterial genera were Lactiplantibacillus (66.79%), Secundilactobacillus (15.12%), Pediococcus (7.50%), Marinilactibacillus (4.92%), and Enterobacter (3.88%), while most common fungal genera were Saccharomyces (63.13%), Bannoa (11.49%), Candida (8.34%), Wickerhamomyces (4.15%), and Kluyveromyces (2.04%). Treatments also affected microbial diversity in biofilms. Adding DMDC significantly reduced the number of bacterial taxa compared to untreated olives. Additionally, the Spanish-style processing resulted in lower bacterial diversity. In contrast, fungi displayed an opposite trend, with the highest similarity found in naturally processed olives. CoDA revealed that the bacterial genera Marinilactibacillus and Lactiplantibacillus were characteristic of Spanish-style olives, while Secundilactobacillus and Pediococcus were associated with naturally processed olives. For fungi, Saccharomyces was related to both Spanish-style and naturally processed olives. The results indicate that the treatments influenced bacterial and fungal populations in brines and olive biofilms and their relationships. These effects may help modulate/control the microbial evolution during olive fermentation.

Efficacy of orange terpene against Escherichia coli biofilm on beef and food contact surfaces

Foodborne pathogen Escherichia coli frequently causes foodborne infections. In our study, we investigated the antibiofilm activity of orange terpene (OT) against E. coli biofilms on a food surface (beef) and different surfaces that come into touch with food, including stainless steel (SS), polyethylene terephthalate (PET), low-density polyethylene (LDPE), and rubber (SR). The study findings revealed that OT significantly (P < 0.05) eliminated 48-h-old biofilms from all food contact surfaces (SS: 2.09 log CFU/cm2, PET: 1.95 log CFU/cm2, LDPE: 1.94 log CFU/cm2, and SR: 1.4 log CFU/cm2). Additionally, on beef surfaces, OT at a minimum inhibitory concentration (MIC) of 0.13 % demonstrated the ability to inhibit biofilm development by approximately 1.5 log CFU/cm2 and reduced pre-formed biofilms by 2.02 log CFU/cm2. Our sensory evaluations showed that it had no adverse impacts on beef color and texture, although it slightly altered the natural odor of beef. Quantitative and qualitative assessments showed that OT has strong bactericidal actions on biofilm populations. It significantly altered cell surface hydrophobicity, reduced cellular ATP levels, and inhibited cell auto-aggregation in planktonic cells (P < 0.05). As a result, our findings emphasize the antibacterial potentiality of OT in reducing the biofilm of E. coli in the food sector.

Assessment of the Antibiofilm Performance of Silver-Containing Wound Dressings: A Dual-Species Biofilm Model.

Background It is commonly accepted that microorganisms found within hard-to-heal wounds are present in biofilm form. Biofilms are often polymicrobial in nature, which increases their virulence and tolerance to antimicrobial agents. The aim of this study was to compare the antibiofilm activity of silver-containing antimicrobial wound dressings in a dual-species simulated wound biofilm model. Materials and methods Four silver-containing wound dressings were evaluated in vitro: Aquacel® Ag+ Extra™ dressing, KerraContact® Ag dressing, Durafiber* Ag dressing, and UrgoClean Ag dressing. Each dressing was applied to a simulated wound assembly containing biofilm-gauze inoculated with Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). Each biofilm-inoculated gauze was incubated at 35±3ºC for 6, 24, 48 and 72 hours. Enumeration of surviving biofilm bacteria at each time point was performed in triplicate for each test dressing and its equivalent control. Results Aquacel® Ag+ Extra™ dressing was observed to reduce the biofilm population within 24 hours with a >4 log10 kill observed for K. pneumoniae and >6 log10 for MRSA from an initial biofilm challenge of 4.16×109 CFU/mL. This kill rate was sustained for the duration of the challenge period, with Aquacel®Ag+ Extra™ dressing reducing the biofilm population to non-detectable levels (<30 Colony Forming Units (CFU) per test) by 72 hours for K. pneumoniae and by 48 hours for MRSA. KerraContact® Ag dressing demonstrated an initial reduction at 6 hours of ~2 log10 in both K. pneumoniae and MRSA. Durafiber* Ag dressing exhibited a slight, gradual reduction in biofilm population over the course of the test period, reducing each challenge organism by ~2.5 log10 by 72 hours. UrgoClean Ag was shown to have little to no impact on the dual-species biofilm with levels remaining similar or greater than that recovered prior to dressing application. The no-dressing biofilm-colonised gauze control demonstrated that the biofilm bacteria remained viable throughout the test period and species population proportionality was maintained. Conclusion Using a dual-species simulated wound biofilm model comprising the pathogens K. pneumoniae and MRSA, Aquacel® Ag+ Extra™ dressing demonstrated significantly greater antibiofilm activity than the other silver-containing dressings. The enhanced antibiofilm activity of Aquacel® Ag+ Extra™ dressing in this study may be attributed to the additional antibiofilm agents, ethylenediaminetetraacetic acid and benzethonium chloride, contained within the dressing.

Dynamics of Nakaseomyces glabratus biofilm formation

Formation of biofilms of microorganisms, including those of Nakaseomyces glabratus, is responsible for the development of local and systemic pathologies in humans and animals. The system of gene expression coordination (quorum sensing) in the representation of signaling molecules allows regulation of the amount and composition of biofilm populations thus expanding the adaptive capacity of microorganisms. In the presence of gingivitis and odontolithiasis clinical signs in dogs, excessive growth of gram-positive yeast microorganisms is a differential sign of the decreased resistance of the digestive system mucous membranes to colonization. Examination of the densitometric and morphometric parameters revealed general patterns of biofilm formation, regardless of the source of Nakaseomyces glabratus isolates. Depending on the time of cultivation of the microorganisms, a gradual increase in the optic density absolute values was established. Intercellular communications were achieved by coaggregation of the heteromorphic structures, which formed clusters with rounded liquid-containing formations detected among them. The population immobilization of the architectonics of the mature three-dimensional biofilm, as consistent with cultivation conditions, was accompanied by the differentiation of numerous cells of different sizes and shapes depending on the stage of the cell cycle. Results of the examination of the general patterns of the heterogeneous micromycete population development are promising for expanding the boundaries of knowledge of the adaptation mechanisms of ubiquitous microorganisms to long-term in vivo and in vitro persistence. Methods for studying morphometric and densitometric indicators avoiding interfering into the natural biofilm architectonics are recommended to optimize the long-term and retrospective mycological studies, as well as to develop effective mycosis treatment and prevention regimens.

Impact of surface topography and hydrodynamic flow conditions on single and multispecies biofilm formation by Escherichia coli O157:H7 and Listeria monocytogenes in presence of promotor bacteria

Biofilm formation of E. coli O157:H7 and L. monocytogenes in the presence of promotor bacteria R. insidiosa was evaluated on equipment surfaces under different hydrodynamic flow conditions in a Centers for Disease Control and Prevention biofilm reactor (CBR). The CBR containing 330 ml bacterial suspension (6 log CFU/ml in 10% TSB) was used to grow the biofilms on SS 316 L, PTFE, Polycarbonate and EPDM rubber surface coupons under 100, 200 and 300 rpm (shear stresses 0.368, 1.218 and 2.462 N/m2, respectively) for 24 h in batch flow and additional 24 h in continuous flow conditions. Surface roughness and topography of coupons were measured by electron microscopy. Bacterial populations in biofilms were determined by scrapping of coupon surfaces and plating on selective media. Multispecies E. coli O157:H7 biofilm formations were affected by the surface and shear stress. SS, PTFE, and PC exhibited a significant increased biofilm growth at higher shear stress of 2.462 N/m2 compared to 0.368 N/m2. Multispecies L. monocytogenes biofilms formation decreased with increasing shear stress on all the surface materials. Our study emphasizes the importance of surface skewness and kurtosis besides surface's roughness while selecting the material for food processing environments.

Unveiling the microbial diversity of biofilms on titanium surfaces in full-scale water-cooling plants using metagenomics approach.

Microbial colonization on the titanium condenser material (TCM) used in the cooling system leads to biofouling and corrosion and influences the water supply. The primary investigation of the titanium condenser was infrequently studied on characterizing biofilm-forming bacterial communities. Different treatment methods like electropotential charge, ultrasonication, and copper coating of titanium condenser material may influence the microbial population over the surface of the titanium condensers. The present study aimed to catalog the primary colonizers and the effect of different treatment methods on the microbial community. CFU (1.7 × 109CFU/mL) and ATP count (< 5000 × 10-7 relative luminescence units) showed a minimal microbial population in copper-coated surface biofilm as compared with the other treatments. Live and dead cell result also showed consistency with colony count. The biofilm sample on the copper-coated surface showed an increased dead cell count and decreased live cells. In the metagenomic approach, the microbiome coverage was 10.06Mb in samples derived from copper-coated TCM than in other treated samples (electropotential charge-17.94Mb; ultrasonication-20.01Mb), including control (10.18Mb). Firmicutes preponderate the communities in the biofilm samples, and Proteobacteria stand next in the population in all the treated condenser materials. At the genus level, Lactobacillaceae and Azospirillaceae dominated the biofilm community. The metagenome data suggested that the attached community is different from those biofilm samples based on the environment that influences the bacterial community. The outcome of the present study depicts that copper coating was effective against biofouling and corrosion resistance of titanium condenser material for designing long-term durability.

Antibacterial and anti-biofilm activities of antibiotic-free phosphatidylglycerol/docosahexaenoic acid lamellar and non-lamellar liquid crystalline nanoparticles

Infectious diseases, particularly those associated with biofilms, are challenging to treat due to an increased tolerance to commonly used antibiotics. This underscores the urgent need for innovative antimicrobial strategies. Here, we present an alternative simple-by-design approach focusing on the development of biocompatible and antibiotic-free nanocarriers from docosahexaenoic acid (DHA) that has the potential to combat microbial infections and phosphatidylglycerol (DOPG), which is attractive for use as a biocompatible prominent amphiphilic component of Gram-positive bacterial cell membranes. We assessed the anti-bacterial and anti-biofilm activities of these nanoformulations (hexosomes and vesicles) against S. aureus and S. epidermidis, which are the most common causes of infections on catheters and medical devices by different methods (including resazurin assay, time-kill assay, and confocal laser scanning microscopy on an in vitro catheter biofilm model). In a DHA-concentration-dependent manner, these nano-self-assemblies demonstrated strong anti-bacterial and anti-biofilm activities, particularly against S. aureus. A five-fold reduction of the planktonic and a four-fold reduction of biofilm populations of S. aureus were observed after treatment with hexosomes. The nanoparticles had a bacteriostatic effect against S. epidermidis planktonic cells but no anti-biofilm activity was detected. We discuss the findings in terms of nanoparticle-bacterial cell interactions, plausible alterations in the phospholipid membrane composition, and potential penetration of DHA into these membranes, leading to changes in their structural and biophysical properties. The implications for the future development of biocompatible nanocarriers for the delivery of DHA alone or in combination with other anti-bacterial agents are discussed, as novel treatment strategies of Gram-positive infections, including biofilm-associated infections.

Substrate geometry affects population dynamics in a bacterial biofilm

Biofilms inhabit a range of environments, such as dental plaques or soil micropores, often characterized by noneven surfaces. However, the impact of surface irregularities on the population dynamics of biofilms remains elusive, as most experiments are conducted on flat surfaces. Here, we show that the shape of the surface on which a biofilm grows influences genetic drift and selection within the biofilm. We culture Escherichia coli biofilms in microwells with a corrugated bottom surface and observe the emergence of clonal sectors whose size corresponds to that of the corrugations, despite no physical barrier separating different areas of the biofilm. The sectors are remarkably stable and do not invade each other; we attribute this stability to the characteristics of the velocity field within the biofilm, which hinders mixing and clonal expansion. A microscopically detailed computer model fully reproduces these findings and highlights the role of mechanical interactions such as adhesion and friction in microbial evolution. The model also predicts clonal expansion to be limited even for clones with a significant growth advantage-a finding which we confirm experimentally using a mixture of antibiotic-sensitive and antibiotic-resistant mutants in the presence of sublethal concentrations of the antibiotic rifampicin. The strong suppression of selection contrasts sharply with the behavior seen in range expansion experiments in bacterial colonies grown on agar. Our results show that biofilm population dynamics can be affected by patterning the surface and demonstrate how a better understanding of the physics of bacterial growth can be used to control microbial evolution.

Common food preservatives impose distinct selective pressures on Salmonella Typhimurium planktonic and biofilm populations

Food preservatives are crucial in controlling microbial growth in processed foods to maintain food safety. Bacterial biofilms pose a threat in the food chain by facilitating persistence on a range of surfaces and food products. Cells in a biofilm are often highly tolerant of antimicrobials and can evolve in response to antimicrobial exposure. Little is known about the efficacy of preservatives against biofilms and their potential impact on the evolution of antimicrobial resistance. In this study we investigated how Salmonella enterica serovar Typhimurium responded to subinhibitory concentrations of four food preservatives (sodium chloride, potassium chloride, sodium nitrite or sodium lactate) when grown planktonically and in biofilms. We found that each preservative exerted a unique selective pressure on S. Typhimurium populations. There was a trade-off between biofilm formation and growth in the presence of three of the four preservatives, where prolonged preservative exposure resulted in reduced biofilm biomass and matrix production over time. All three preservatives selected for mutations in global stress response regulators rpoS and crp. There was no evidence for any selection of cross-resistance to antibiotics after preservative exposure. In conclusion, we showed that preservatives affect biofilm formation and bacterial growth in a compound specific manner. We showed trade-offs between biofilm formation and preservative tolerance, but no antibiotic cross-tolerance. This indicates that bacterial adaptation to continuous preservative exposure, is unlikely to affect food safety or contribute to antibiotic resistance.

Optimization of an in vitro Pseudomonas aeruginosa Biofilm Model to Examine Antibiotic Pharmacodynamics at the Air-Liquid Interface

Pseudomonas aeruginosa is an important cause of lower respiratory tract infections, such as ventilator-associated bacterial pneumonia (VABP). Using inhaled antibiotics to treat VABP can achieve high drug concentrations at the infection site while minimizing systemic toxicities. Despite the theoretical advantages, clinical trials have failed to show a benefit for inhaled antibiotic therapy in treating VABP. A potential reason for this discordance is the presence of biofilm-embedded bacteria in lower respiratory tract infections. Drug selection and dosing are often based on data from bacteria grown planktonically. In the present study, an in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model was optimized to evaluate the activity of simulated epithelial lining fluid exposures of inhaled and intravenous doses of polymyxin B and tobramycin against two P. aeruginosa strains. Antibiotic activity was also determined against the P. aeruginosa strains grown planktonically. Our study revealed that inhaled antibiotic exposures were more active than their intravenous counterparts across biofilm and planktonic populations. Inhaled exposures of polymyxin B and tobramycin exhibited comparable activity against planktonic P. aeruginosa. Although inhaled polymyxin B exposures were initially more active against P. aeruginosa biofilms (through 6 h), tobramycin was more active by the end of the experiment (48 h). Together, these data slightly favor the use of inhaled tobramycin for VABP caused by biofilm-forming P. aeruginosa that are not resistant to either antibiotic. The optimized in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model may be beneficial for the development of novel anti-biofilm agents or to optimize antibiotic dosing for infections such as VABP.

Vancomycin-Polyguanidino Dendrimer Conjugates Inhibit Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria and Eradicate Biofilm-Associated S.aureus.

The global challenge of antibiotic resistance necessitates the introduction of more effective antibiotics. Here we report a potentially general design strategy, exemplified with vancomycin, that improves and expands antibiotic performance. Vancomycin is one of the most important antibiotics in use today for the treatment of Gram-positive infections. However, it fails to eradicate difficult-to-treat biofilm populations. Vancomycin is also ineffective in killing Gram-negative bacteria due to its inability to breach the outer membrane. Inspired by our seminal studies on cell penetrating guanidinium-rich transporters (e.g., octaarginine), we recently introduced vancomycin conjugates that effectively eradicate Gram-positive biofilm bacteria, persister cells and vancomycin-resistant enterococci (with V-r8, vancomycin-octaarginine), and Gram-negative pathogens (with V-R, vancomycin-arginine). Having shown previously that the spatial array (linear versus dendrimeric) of multiple guanidinium groups affects cell permeation, we report here for the first time vancomycin conjugates with dendrimerically displayed guanidinium groups that exhibit superior efficacy and breadth, presenting the best activity of V-r8 and V-R in single broad-spectrum compounds active against ESKAPE pathogens. Mode-of-action studies reveal cell-surface activity and enhanced vancomycin-like killing. The vancomycin-polyguanidino dendrimer conjugates exhibit no acute mammalian cell toxicity or hemolytic activity. Our study introduces a new class of broad-spectrum vancomycin derivatives and a general strategy to improve or expand antibiotic performance through combined mode-of-action and function-oriented design studies.

PCV13 vaccine prevents pneumococcal biofilms without affecting Staphylococcus aureus population within the polymicrobial biofilm.

In respiratory pathogens such as Streptococcus pneumoniae, biofilm formation is associated with the colonization of the nasopharynx and chronic respiratory infection. Previous data have shown that pneumococcal conjugate vaccines (PCVs) had an impact on S. pneumoniae colonization and a potential replacement by other respiratory pathogens such as Staphylococcus aureus. The objective of this work was to evaluate the evasion of the immune system by monospecific biofilms and by S. aureus-S. pneumoniae mixed biofilms. We performed opsonophagocytosis assays (OPA) using human HL-60 against previously disaggregated monospecific biofilms of MSSA, MRSA and S. aureus-S. pneumoniae mixed biofilms. We used pre-immune and post-immune serum from immunocompetent adult patients vaccinated with PCV13. Immune sera had a clear effect in reducing pneumococcal biofilms of serotypes 3, 14, 18C, 19F and 19A, whereas had no effect in non-PCV13 serotypes such as 8, 11A and 24F. Our study confirmed that serum from vaccinated patients with PCV13 did not have any effect in reducing S. aureus population in monospecific biofilms, regardless the methicillin resistance phenotype. Moreover, immunized sera from vaccinated patients with PCV13 did not have any effect in S. aureus population in the mixed biofilm, whereas significantly reduced the population of pneumococcal serotype 19A strain in the mixed biofilm which is of great interest because this serotype is included in PCV13, and it is associated with vaccine failures.

A Novel Inhibitor against the Biofilms of Non-Tuberculous Mycobacteria.

Non-tuberculous Mycobacteria (NTM), previously classified as environmental microbes, have emerged as opportunistic pathogens causing pulmonary infections in immunocompromised hosts. The formation of the biofilm empowers NTM pathogens to escape from the immune response and antibiotic action, leading to treatment failures. NF1001 is a novel thiopeptide antibiotic first-in-class compound with potent activity against planktonic/replicating and biofilm forms of various NTM species. It is potent against both drug-sensitive and -resistant NTM. It has demonstrated a concentration-dependent killing of replicating and intracellularly growing NTM, and has inhibited and reduced the viability of NTM in biofilms. Combination studies using standard-of-care (SoC) drugs for NTM exhibited synergetic/additive effects, but no antagonism against both planktonic and biofilm populations of Mycobacterium abscessus and Mycobacterium avium. In summary, the activity of NF1001 alone or in combination with SoC drugs projects NF1001 as a promising candidate for the treatment of difficult-to-treat NTM pulmonary diseases (NTM-PD) and cystic fibrosis (CF) in patients.

Геномные перестройки влияют на устойчивость биопленок почвенных бактерий Azospirillum brasilense к абиотическим стрессам

The bacteria Azospirillum brasilense, used as biofertilizers, have a significant positive effect on the growth and development of plants. The genome of the strain A. brasilense Sp7 is represented by a chromosome and numerous plasmids with molecular weight of 90, 115, and over 300 MDa. Genomic rearrangements that cause changes in the “plasmid profile” can contribute to the formation of subpopulations or phenotypic variants in a bacterial population. There is little data on the role of such rearrangements in the adaptation of A. brasilense to dynamic environmental conditions. The ability of azospirilla to form biofilms also has a determined significance for the successful functioning of the plant-microbial association and the resistance of bacteria and plants to various abiotic stresses. The purpose of this work consisted of the analysis of the genomic rearrangements in spontaneous derivatives of A. brasilense Sp7 and the assessment of the resistance of their biofilms to drying, water stress and oxidative stress. PCR analysis to detect changes in the structure of genomic DNA was performed using primers corresponding to known conservative motifs in repetitive bacterial nucleotide sequences. The relative amount of the biofilm biomass was assessed by measuring the crystal violet A540 desorbed after staining. The level of relative respiratory activity of cells in biofilms was determined by the fluorometric resazurin test. The non-penetrating osmotic agent PEG 6000 was used to create the osmotic/water stress model. It was shown that rearrangements in genomic DNA contribute to the formation of stable phenotypic variants of the Sp7 strain, which form biofilms in different ways under water stress conditions. A derived strain of A. brasilense Sp7.8, the biofilm population of which is more resistant to water stress compared to the parent strain was selected.

Antibacterial and antibiofilm effects of gold and silver nanoparticles against the uropathogenic &lt;i&gt;Escherichia coli&lt;/i&gt; by scanning electron microscopy (SEM) analysis

Uropathogenic Escherichia coli (UPEC) is a nosocomial pathogen associated with urinary tract infections and biofilm formation, which contributes to antibiotic resistance. Discovering potent antibacterial agents is crucial. This study aimed to assess the antibacterial and antibiofilm effects of gold and silver nanoparticles on UPEC using Scanning Electron Microscopy (SEM). UPEC biofilms were cultivated on nitrocellulose membranes for 48 hours at 37°C, then treated with gold nanoparticles (50 ppm and 100 ppm) and silver nanoparticles (50 ppm and 100 ppm) for another 48 hours. Antibacterial and antibiofilm activities were evaluated through cell density and SEM analysis. SEM revealed lower cell density, reduced biofilm formation, and altered cell morphology with rough, wrinkled surfaces after nanoparticle treatment. In conclusion, gold and silver nanoparticles exhibit antibacterial and antibiofilm properties, as observed in SEM analysis. SEM is a valuable tool for studying the antimicrobial effects of nano gold and silver on bacterial cell morphology and biofilm populations.

Bactericidal effect of Iberin combined with photodynamic antimicrobial chemotherapy against Pseudomonas aeruginosa biofilm cultured on ex vivo wound model

Wounds infected by Pseudomonas aeruginosa (P. aeruginosa) biofilms are characterized by poor healing and by being long lasting. Pyocyanin and pyoverdine are exotoxins that contribute to P. aeruginosa pathogenicity in wound infections and are known as virulence factors. Despite the usefulness of antimicrobial photodynamic therapy (PDT) in the management of wound infections, biofilms are hurdle for microbial photoinactivation. Quorum sensing (QS) is a cell density-dependent chemical signaling system P. aeruginosa uses to regulate biofilm formation and virulence factors production. In the current study, QS attenuation was used in combination with PDT against P. aeruginosa biofilm cultured on skin explant. Iberin is a QS inhibitor that attenuates P. aeruginosa virulence and affects biofilm integrity. The antibiofilm and QS inhibitory activities of iberin in combination with either riboflavin or 5,10,15,20-Tetrakis(1-methyl-4-pyridinio) porphyrin tetra p-toluenesulfonate (TMP) mediated PDT were investigated using viable count method and pyocyanin and pyoverdine assays, respectively.No bactericidal activity was reported when iberin was added to a mature biofilm (24 h) followed by PDT. When added to a growing biofilm at multiple time points (0 h, 24 h and 48 h), iberin inhibited P. aeruginosa biofilm QS signaling system. This inhibitory effect resulted in an observable decrease in the levels of the QS-regulated virulence factors, pyocyanin and pyoverdine, without any effect on the growth of the biofilm cultures. These changes in biofilm virulence were associated with a decrease in biofilm resistance to PDT and caused bactericidal effect upon photosensitizers treatment and irradiation. Iberin-treated-riboflavin-mediated PDT resulted in a significant 1.3 log reduction in biofilm population. Similarly, iberin-treated-TMP-mediated PDT caused a significant 1.8 log reduction in biofilm population. The combination of QS inhibitor with PDT is a promising alternative antimicrobial therapy for the management of biofilms.

Transcriptional profiling and genetic analysis of a cystic fibrosis airway-relevant model shows asymmetric responses to growth in a polymicrobial community.

Bacterial infections in the lungs of persons with cystic fibrosis are typically composed of multispecies biofilm-like communities, which modulate clinically relevant phenotypes that cannot be explained in the context of a single species culture. Most analyses to date provide a picture of the transcriptional responses of individual pathogens; however, there is relatively little data describing the transcriptional landscape of clinically relevant multispecies communities. Harnessing a previously described cystic fibrosis-relevant, polymicrobial community model consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, we performed an RNA-Seq analysis on the biofilm population to elucidate the transcriptional profiles of the community grown in artificial sputum medium (ASM) as compared to growth in monoculture, without mucin, and in fresh medium supplemented with tobramycin. We provide evidence that, although the transcriptional profile of P. aeruginosa is community agnostic, the transcriptomes of S. aureus and S. sanguinis are community aware. Furthermore, P. aeruginosa and P. melaninogenica are transcriptionally sensitive to the presence of mucin in ASM, whereas S. aureus and S. sanguinis largely do not alter their transcriptional profiles in the presence of mucin when grown in a community. Only P. aeruginosa shows a robust response to tobramycin. Genetic studies of mutants altered in community-specific growth provide complementary data regarding how these microbes adapt to a community context. IMPORTANCE Polymicrobial infections constitute the majority of infections in the cystic fibrosis (CF) airway, but their study has largely been neglected in a laboratory setting. Our lab previously reported a polymicrobial community that can help explain clinical outcomes in the lungs of persons with CF. Here, we obtained transcriptional profiles of the community versus monocultures to provide transcriptional information about how this model community responds to CF-related growth conditions and perturbations. Genetic studies provide complementary functional outputs to assess how the microbes adapt to life in a community.

Influence of Central Metabolism Disruption on Escherichia coli Biofilm Formation.

Biofilms are widely recognized as a prominent mode of microbial growth and strategy of antimicrobial tolerance in many environments. Characteristics that are often overlooked in biofilm investigations include the examination of metabolic pathways as the assumption might be that interference with central pathways such as glycolysis would only reduce growth and thus not be meaningful. Using the Keio collection of Escherichia coli mutants, we investigated the influence of biofilm formation and planktonic growth in full strength and diluted Luria-Bertani (LB) broth using strains with a disruption of glycolysis (Δpgi), the Entner-Doudoroff pathway (Δedd), or the pentose phosphate pathway (Δgnd). Unexpectedly, in contrast to the E. coli Keio parent strain (BW25113), planktonic growth was enhanced in full strength and diluted LB broth in the metabolic mutants. Using a microtiter biofilm assay, the E. coli parent strain showed the highest crystal violet staining. However, when analyzed by culture assays, there was an increase in biofilm populations in the mutants in comparison to the parent strain. Fluorescence microscopy showed differences in colonization patterns in the strains. Given the availability of mutant collections in many model organisms, similar metabolic studies are warranted for biofilms, given their importance in nature.

Single cells and bacterial biofilm populations in chronic wound infections.

Chronic wounds and chronic ulcers are an increasing problem associated with high health care burden and patient burden. The arrested healing of chronic wounds has, in part, been attributed to the presence of biofilms. Substantial research has documented the presence of biofilms in chronic wounds, and many mechanisms of host-pathogen interactions have been uncovered to explain the arrested healing. However, the paradigm of whether biofilms are only observed in chronic infections was recently challenged when biofilms were also observed in acute infections. Here, we characterize the distribution of bacteria in lower leg wounds with particular emphasis on Pseudomonas aeruginosa and Staphylococcus aureus by confocal laser scanning microscopy combined with PNA-FISH staining and routine culture of bacteria. We show that 40% of wounds contained either P. aeruginosa or S. aureus biofilms and demonstrate the presence of scattered single cells in tissues stained with a universal bacterial PNA-FISH probe. Thus, we demonstrate that chronic wounds do not only harbor bacteria organized in biofilms, but also carry populations of scattered single cells and small cell clusters of only a few bacteria. Our findings may influence diagnostic tools being developed to only target biofilms, where single-cell subpopulations thus may be overlooked and possibly lead to false-negative results.